Eukaryotic communities associated with the decomposition of rice straw compost in a Japanese rice paddy field estimated by DGGE analysis

To estimate the succession and phylogenetic composition of the eukaryotic communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analysis targeting 18S rDNA followed by sequencing was conducted in a Japanese paddy field. The eukaryotic communities in rice straw compost incorporated into the flooded paddy field were influenced by the mid-season drainage and mainly composed of fungi (Ascomycota, Zygomycota, and Chytridiomycota) and protozoa (Ciliophora, Euglyphida, and Dactylopodida), most of which existed continuously during the cultivation period of paddy rice. The results indicated that these eukaryotic members were associated with the decomposition of rice straw compost in paddy field soil directly or indirectly.     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.     

稻田冬种绿肥对水稻产量和土壤肥力的影响

为探讨冬季种植绿肥作物改良土壤的生态作用, 以冬闲连作为对照, 通过连续8年的定位试验研究了冬种绿肥(紫云英、油菜和黑麦草)收获后翻压还田对后作水稻产量及土壤肥力的影响。结果表明: 连续8年冬种绿肥期间, 冬种绿肥各处理的水稻平均产量均高于冬闲对照, 但不同处理增产效果有所不同, 其中紫云英 早稻 晚稻处理增产效果最明显, 为10.48%, 其次是油菜 早稻 晚稻处理(7.31%), 黑麦草 早稻 晚稻处理的增产效果较差, 仅为5.76%。紫云英 早稻 晚稻处理的土壤pH和碱解氮含量最高, 与其他处理相比, pH增加0.58%~1.37%, 碱解氮增加6.59%~35.35%。黑麦草 早稻 晚稻处理的土壤有机质、有效磷、速效钾含量和C/N在各处理中最高, 分别较其他处理高2.99%~4.98%、19.25%~31.05%、16.97%~22.15%和0.97%~6.61%。相关性分析结果表明, 水稻产量与每穗颖花数、结实率、千粒重及土壤碱解氮、有效磷极显著相关(P<0.01), 与每穗粒数、有机质、速效钾显著相关(P<0.05), 而与穗长、有效穗数及土壤pH和C/N相关不显著; 水稻产量各构成要素与土壤养分之间的关系极为密切。综合来看, 稻田冬种绿肥有利于增加水稻产量和改善土壤肥力。     

Phylogenetic study on a bacterial community in the floodwater of a Japanese paddy field estimated by sequencing 16S rDNA fragments after denaturing gradient gel electrophoresis

Phylogenetic positions of characteristic bands of 16S rDNA that were obtained from the floodwater of a Japanese paddy field by denaturing gradient gel electrophoresis (DGGE) analysis in a previous work (Biol Fertil Soils 36:306–312, 2002) were determined to identify dominant bacterial members in the floodwater. Sequences of DGGE bands were affiliated with the Cytophaga–Flavobacterium–Bacteroides group, β-Proteobacteria, and Actinobacteria and showed phylogenetically close relationships with species inhabiting other aquatic environments, although most of their closest relatives were uncultured bacterial clones.     

Succession and phylogenetic profile of eukaryotic communities in the composting process of rice straw estimated by PCR-DGGE analysis

The succession and phylogenetic profile of eukaryotic communities during the composting process of rice straw (RS) were studied by applying polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 18S rDNA. Principal component analysis and cluster analysis of the DGGE band patterns of eukaryotic communities resulted in exactly the same grouping as found with phospholipid fatty acid (PLFA) analysis (Cahyani et al. in Soil Sci Plant Nutr 48:735, 2002) and by the DGGE pattern analysis of the bacterial communities (Cahyani et al. in Soil Sci Plant Nutr 49:619, 2003) for the same samples, namely the communities characterizing the pre-composting stage (initial RS materials), and thermophilic, middle, and curing stages of the compost. Different eukaryotic members characterized the respective stages as follows: fungi (Ascomycota) for the initial RS materials, protozoans (Apicomplexa) as well as the fungi (Ascomycota) of the initial RS materials for the thermophilic stage, fungi (Ascomycota and Basidiomycota), protozoans (Opalozoa, Ciliophora and Leptomyxida), nematodes and stramenopiles for the middle stage, and fungi (Ascomycota, Zygomycota and Oomycota), algae (Haptophyceae and Chrysophyceae), and nematodes for the curing stage, respectively. Temperature, moisture content, and substrates available seemed to play a key role in determining the composition of eukaryotic members present at the respective stages of the composting process of RS.     

Microbial community responsible for the decomposition of rice straw in a paddy field: estimation by phospholipid fatty acid analysis

To estimate the microbial communities responsible for rice straw decomposition in paddy field, phospholipid fatty acid (PLFA) composition of leaf sheaths and blades was analyzed during the decomposition of both residues under upland conditions after harvest and under flooded conditions at the time of transplanting of rice plants. In addition, rice straw that had been placed in the field under upland conditions (November to April) was taken out in spring, and placed again in the same field under flooded conditions at the time of transplanting. High proportions of the branched-chain PLFAs were observed under flooded conditions (June to September); the proportions of straight mono-unsaturated and straight poly-unsaturated PLFAs were high under upland conditions in the winter season for 4 months. The dominant PLFAs in straight mono-unsaturated, straight poly-unsaturated and branched-chain PLFA groups were 18:19, 18:17 and 16:17c, 18:26c and i15:0, i17:0 and ai15:0, respectively, under both upland and flooded conditions. These findings indicated the important roles of Gram-negative bacteria and fungi under upland conditions and of Gram-positive bacteria and anaerobic Gram-negative bacteria under flooded conditions. Cluster analysis of PLFA composition showed the difference of community structure of microbiota in rice straw between upland and flooded conditions. In addition principal component analysis revealed the difference between leaf sheaths and blades under upland conditions and indicated that the content of straight unsaturated PLFAs (sheaths > blades) characterized their community structures.     

Incorporation of C-labeled rice-straw-derived carbon into microbial communities in submerged rice field soil and percolating water

Rice straw is a major organic material applied to rice fields. The microorganisms growing on rice-straw-derived carbon have not been well studied. Here, we applied ¹³C-labeled rice straw to submerged rice soil microcosms and analyzed phospholipid fatty acids (PLFAs) in the soil and percolating water to trace the assimilation of rice-straw-derived carbon into microorganisms. PLFAs in the soil and water were markedly enriched with ¹³C during the first 3 days of incubation, which indicated immediate incorporation of rice-straw-derived carbon into microbial biomass. The enrichment of PLFAs in the percolating water with ¹³C suggested that microorganisms other than the population colonizing rice straw also assimilated rice-straw-derived carbon or that some bacterial groups were selectively released from the straw. The microbial populations could be categorized into two communities based on the carbon isotope data of the PLFAs: those derived from rice straw and those derived from soil organic matter (SOM). The composition of the PLFAs from the two communities differed, which indicated the assimilation of rice-straw-derived carbon by a subset of microbial populations. The composition of rice-straw-derived PLFAs in the percolating water was also distinct from that in the soil.     

cry1Ac/cpti转基因大米水稻土中微生物群落的多样性研究

Two types of cry1Ac/cpti transgenic rice(GM1 and GM2)and their parental non-cry1Ac/cpti rice(CK1 and CK2)were planted in the field at Wufeng,Fujian Province,China for four years to investigate the influence of genetically modified rice on diversity of bacterial and fungal community in the paddy soil.The community composition and abundance of bacteria or fungi in the paddy soil were assessed at different growth stages of rice by denaturing gradient gel electrophoresis and real-time polymerase chain reaction based on 16S rRNA gene or SSU rRNA gene in the 4th year after the experimental establishment.The composition of bacterial or fungal community changed during rice growth,while no significant differences were observed between the fields cultivated with GM1and CK1,or between the fields cultivated with GM2 and CK2 in either bacterial or fungal community composition.The copy numbers of bacterial 16S rRNA gene in the soils with CK1,CK2,GM1 and GM2 ranged from 5.64×1011to 6.89×1011copies g-1dry soil at rice growth stages,and those of fungal SSU rRNA gene from 5.24×108to 8.68×108copies g-1dry soil.There were no marked differences in the copies of bacterial 16S rRNA gene or fungal SSU rRNA gene between CK1 and GM1 or between CK2 and GM2at any growth stage of rice.Planting cry1Ac/cpti transgenic rice had no significant effect on composition and abundance of bacterial and fungal community in paddy soil during the rice growing season at least in the short term.     

土壤改良剂对冷浸田土壤特性和水稻群体质量的影响

以南方典型冷浸田为研究对象, 在明沟排水的基础上, 通过田间定位试验, 以不施土壤改良剂为对照, 研究了施用不同土壤改良剂(自研的脱硫灰改良剂、生物活性炭, 市售的土壤改良剂石灰、硅钙肥、腐植酸)对冷浸田氧化还原电位、土壤呼吸强度、土壤微生物数量、水稻群体构建及产量构成因素的影响。结果表明, 施用改良剂能够改善土壤理化性状, 提升土壤速效养分和pH,但除脱硫灰处理外, 其他改良剂处理对土壤Eh未产生显著影响。施用不同土壤改良剂在水稻各生育期均能有效增强土壤微生物呼吸强度和放线菌数量, 并且放线菌数量达到差异性显著水平(P<0.05), 生物活性炭处理下土壤呼吸强度和放线菌数量分别较对照增加67.6%和127.6%。各土壤改良剂处理与CK相比较均有助于提高叶片SPAD、茎蘖数、水稻干物质积累量、成穗数、穗粒数、产量结实率和根系伤流速率。其中以脱硫灰和生物活性炭处理改良效果最佳, 抽穗后29 d时,根系伤流速率较CK分别提高45.4%和39.1%, 叶片SPAD分别增加27.4%和22.5%; 成熟期水稻成穗数较对照提高12.1%和10.7%,干物质积累量增加68.8%和50.5%,产量分别增加12.8%和10.3%。综上所述, 土壤改良剂可有效改善冷浸田土壤特性及水稻群体质量, 脱硫灰和生物活性炭处理的改良效果最明显, 增产幅度最大。     

Microbial communities responsible for the decomposition of rice straw compost in a Japanese rice paddy field determined by phospholipid fatty acid (PLFA) analysis

To identify the microbial communities responsible for the decomposition of rice straw compost in soil during the rice cultivation period, phospholipid fatty acid (PLFA) composition of rice straw compost was determined by periodically sampling the compost from a Japanese rice field under flooded conditions. About 21% of the compost was decomposed within a period of 3 months. The total amount of PLFAs, as an indicator of microbial biomass, was significantly lower under drained conditions than under flooded conditions and was relatively constant during the flooding period. This indicates that the microbial biomass in the compost samples did not increase during the gradual decomposition of rice straw compost under flooded conditions. The proportion of branched-chain PLFAs (biomarker of Grampositive and anaerobic Gram-negative bacteria) slightly decreased during the early period after placement, and increased gradually afterwards. Among the branched-chain PLFAs, i15:0, ail5:0, i16:0 and i17:0 PLFAs predominated and their proportions increased gradually except for i16:0. The proportion of straight mono-unsaturated PLFAs (biomarker of Gramnegative bacteria) was almost constant throughout the period, and 18:1ω9 and 18:1ω7 PLFAs predominated. The proportion of straight poly-unsaturated PLFAs as a biomarker of eukaryotes including fungi was also constant throughout the period, except for a decrease under drained conditions. Straight poly-unsaturated PLFAs consisted mainly of 18:2ω6c PLFA. Therefore, these results suggest that the proportions of Gram-positive and anaerobic Gram-negative bacteria increased during the decomposition of rice straw compost in flooded paddy field. Statistical analyses enabled to divide PLFA patterns of microbiota in the rice straw compost into two groups, one group consisting of rice straw compost samples collected before mid-season drainage and the other of samples collected after mid-season drainage. Small squared distances among samples in cluster analysis indicated that the community structure of microbiota was similar to each other as a whole. These results suggest that the microbial communities changed gradually during the period of placement, and that mid-season drainage may have affected the community structure of microbiota. Principal component analysis of the PLFA composition suggested that the succession of microbiota along with the decomposition in flooded soil was similar between rice straw compost and rice straw and that the changes in the community structure during the decomposition in flooded soil were more conspicuous for rice straw than for rice straw compost.     

改良剂对反酸田土壤性质与水稻产量的影响

采用盆栽试验研究了不同改良剂在反酸田土壤上的应用效果。结果表明,土壤pH在3.47~4.90范围内,pH与交换性H+和交换性Al3+呈极显著线性负相关关系,而与土壤有效铁和有效硫含量呈显著正相关。不同改良处理在提高作物产量和改良土壤性质方面较对照处理与NPK处理效果明显,且钙镁磷肥、自研改良剂、石灰及碱渣等无机改良剂改良效果显著高于添加腐植酸、精制有机肥、碱性有机肥处理。其改良效果主要体现在提高土壤pH,显著降低土壤交换性H+和交换性Al3+含量,明显增加土壤有效钙和有效镁的含量,改善根系生长环境,增加水稻地上部养分吸收和水稻籽粒产量。综上,初步认为酸害(包括活性酸和潜在酸)是反酸田土壤最主要的限制因子,钙镁磷肥、自研改良剂和石灰等偏碱性且富含有效钙或磷的无机矿物质是农业生产中改良反酸田的适宜改良剂。     

广谱抗真菌蛋白转基因水稻秸秆模拟还田对土壤真菌群落结构的影响

为探讨广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响,本文在室温条件下进行田间秸秆还田模拟试验,设不添加秸秆(S)、添加转基因水稻‘转品1’秸秆(S-Z1)、添加转基因水稻‘转品8’秸秆(S-Z8)、添加非转基因水稻‘七丝软粘’秸秆(S-CK)4个土壤处理,采用传统的平板计数法和变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术,分析广谱抗真菌蛋白转基因水稻秸秆模拟还田过程中土壤可培养真菌数和土壤真菌群落的变化情况。平板计数结果表明,在秸秆降解的第40 d,转基因水稻秸秆处理(S-Z1、S-Z8)与非转基因水稻秸秆处理(S-CK)土壤之间的可培养真菌数差异显著,但秸秆降解中后期(50~90 d),S-Z1、S-Z8和S-CK之间土壤可培养真菌数的差异均不显著。真菌18S r RNA的PCR-DGGE图谱显示,S-Z1、S-Z8和S-CK在秸秆降解过程中没有显著不同的条带出现,仅有个别条带在亮度上存在差异。DGGE图谱条带多样性分析结果表明,在秸秆降解的个别时间段,S-Z1、S-Z8和S-CK之间在丰富度和Shannon-Wiener多样性指数上存在显著差异,而在秸秆降解的整个过程均匀度指数差异均不显著。对DGGE主要条带和差异性条带进行克隆测序后发现,子囊菌占最大比重,其次为担子菌、壶菌,而在转基因和非转基因土壤处理间亮度上存在差异的条带属于子囊菌。以上研究结果表明,广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响是短暂的、不持续的。     

Long-term effects of compost amendment of soil on functional and structural diversity and microbial activity

We studied the effects of applying different composts (urban organic waste, green waste, manure and sewage sludge), mineral fertilizer and compost plus mineral fertilizer on chemical, biological and soil microbiological parameters over a 12‐year period. The organic C and total N levels in soils were increased by all compost and compost + N treatments. Microbial biomass C was significantly (P ≤ 0.05) increased for some compost treatments. In addition, basal respiration and the metabolic quotient (qCO₂) were significantly higher in all soils that had received sewage sludge compost. The Shannon diversity index (H), based on community level physiological profiling, showed a higher consumption of carbon sources in soils treated with compost and compost + N compared with the control. The utilization of different guilds of carbon sources varied amongst the treatments (compost, compost + N or mineral fertilizer). Cluster analysis of polymerase chain reaction‐denaturing gradient gel electrophoresis patterns showed two major clusters, the first containing the mineral fertilization and compost treatments, and the second, the composts + N treatments. No differences in bacterial community structure could be determined between the different types of compost. However, the results suggest that long‐term compost treatments do have effects on the soil biota. The results indicate that the effects on the qCO₂ may be due to shifts in community composition. In this study, it was not possible to distinguish with certainty between the effects of different composts except for compost derived from sewage sludge.     

稻田节肢动物群落对3 种土壤改良剂的响应

试验研究了竹炭、凹凸棒石、木炭3 种土壤改良剂在吸附土壤污染物质、改善土壤物理性状时, 对稻田节肢动物群落的影响。结果表明: 3 种土壤改良剂均显著提高了蜘蛛类个体数量, 特别是优势种八斑球腹蛛(Coleosoma octomaculatum)的个体数量, 竹炭、凹凸棒石处理田八斑球腹蛛的个体数量为对照田的5 倍多,木炭处理田为对照田的4 倍多; 3 种土壤改良剂均显著降低了植食类昆虫的个体数量, 竹炭、凹凸棒石和木炭处理田的目标害虫稻褐飞虱(Nilaparvata lugens)数量分别为对照田的15%、10%和20%; 3 种土壤改良剂均显著提高了蜘蛛类的优势度, 显著降低了植食昆虫类的优势度; 竹炭和凹凸棒石处理的第1 年可显著提高节肢动物群落多样性, 第2 年对节肢动物群落多样性无显著影响, 木炭处理的每年都显著提高了稻田节肢动物群落多样性与复杂度, 增加了稻田生态系统的稳定性。     

Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans,its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis

Abstract

A preliminary survey using 20 conventionally farmed fields in which fumigants have been applied every year showed that the root-lesion nematode Pratylenchus penetrans was distributed both in the upper (0–30?cm) and lower (30–60?cm) soil layers. In six of the 20 fields, P.?penetrans was detected in the lower layers exclusively, suggesting that the most appropriate depth to sample soil is 0–60?cm to estimate the relationship between the density of P.?penetrans and its damage to radish. There was a highly significant correlation (r?=?0.923) between the density of P.?penetrans in the 0–60?cm depth and the number of spots on a radish. No damage to radish was observed in soils with <2.5 individuals of P.?penetrans per 20?g soil before cultivation. However, in cases in which the density of P.?penetrans was 3.4–6.2 individuals per 20?g soil, the number of spots on a radish showed more variation (0–131.5 per radish) and there was no significant correlation between them. The nematode community structure of soils with 3.4–8 individuals of P.?penetrans per 20?g soil, evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis, was significantly different (anova, PC2, P?<?0.05) between soils with low (0–42) and high (more than 80) damage levels, suggesting that radish damage might be predicted on the basis of the prevailing soil nematode community structure.     

稻草还田免耕抛秧对水稻土剖面形态特征的影响

为探讨免耕抛秧栽培对土壤剖面形态特征的影响,分别对连续2年和7年结合稻草还田的常耕和免耕试验的土壤剖面进行了研究。结果表明,常耕对土壤剖面形态特征影响不明显。免耕改变了耕作层亚层的剖面形态特征,且表层土壤疏松和pH值变小;水稻根系向土壤表层集中和裂隙出现部位增高。普通免耕形成的耕作层构型是Aa1-Aa2-Aa3,疏松表层较薄、亚表层有变坚实的趋势。稻草还田免耕形成的耕作层构型是O-Aa1-Aa2-Aa3,鳝血斑的数量增加、土壤颜色加深、疏松和体积质量降低。以稻草还田免耕抛秧形成的土壤剖面协调土壤肥力效果最好。     

Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans, its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis

A preliminary survey using 20 conventionally farmed fields in which fumigants have been applied every year showed that the root-lesion nematode Pratylenchus penetrans was distributed both in the upper (0–30 cm) and lower (30–60 cm) soil layers. In six of the 20 fields, P. penetrans was detected in the lower layers exclusively, suggesting that the most appropriate depth to sample soil is 0–60 cm to estimate the relationship between the density of P. penetrans and its damage to radish. There was a highly significant correlation ( r  = 0.923) between the density of P. penetrans in the 0–60 cm depth and the number of spots on a radish. No damage to radish was observed in soils with <2.5 individuals of P. penetrans per 20 g soil before cultivation. However, in cases in which the density of P. penetrans was 3.4–6.2 individuals per 20 g soil, the number of spots on a radish showed more variation (0–131.5 per radish) and there was no significant correlation between them. The nematode community structure of soils with 3.4–8 individuals of P. penetrans per 20 g soil, evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis, was significantly different ( anova , PC2, P  <   0.05) between soils with low (0–42) and high (more than 80) damage levels, suggesting that radish damage might be predicted on the basis of the prevailing soil nematode community structure.